Enzymatic utilization of cotton oil soap stock

Enzymatic hydrolysis of neutral fat of cotton oil soap stock with a nonspecific lipase produced byOospora lactis F-500 was designed. The culture liquid and a preparation of enzyme obtained by precipitation with isopropanol from a filtrate of the culture liquid were used. Utilization of cotton oil soap stock as the only source of carbon during cultivation of the fungus was studied. The rate of hydrolysis of soap stock fat strongly depended on the way of biological conversion of cotton oil soap stock. The most effective utilization was observed during cultivation of the fungus in the medium containing soap stock.     

Formation of organic acids by the fungus Cladosporium resinae in media containing n-alkanes]

The so-called fungus Cladosporium resinae that often occurs in oil fuels ane increases their acidity grows well at the expense of n-alkanes from C11 to C16. On the n-alkane containing media the fungus grows slowly and only under the stationary conditions. During the fungal cultivation on the media containing n-dodecane or kerosene the culture liquid shows acetic acid and other fatty acids, ketoacids (pyruvic and alpha-ketoglutaric) as well as citric and isocitric acids that dominate among nonvolatile acids. Upon nitrogen deficiency in the medium and comparatively good aeration the content of citric acids increases. The culture liquid of the fungus devoid from the mycelium and nonutilized n-alkanes can be used a a nutrient medium for different microorganisms.     

从米糠油二次皂脚中制取谷维素的研究

本课题的目的是从米糠油二次皂脚中制取谷维素。我们采用新工艺、新技术多相分步结晶,应用于实践,优选条件,谷维素的制取率为米糠油二次皂脚的1.9％,谷维素含量为98％。     

Production of Taxol from Phyllosticta spinarum,an endophytic fungus of Cupressus sp.

Taxol production during the cultivation on a modified liquid and potato dextrose broth medium was indicated for the first time to occur in Phyllosticta spinarum, an endophytic fungus isolated from the needles of Cupressus sp. The presence of taxol in the fungal culture filtrate was confirmed by chromatographic and spectroscopic methods of analysis. The amount of taxol produced by this fungus was quantified by high performance liquid chromatography. The maximum amount of taxol production was obtained in this fungus when grown on M1D medium (235 μg/L) followed by PDB medium (125 μg/L). The results indicate that P. spinarum is an excellent candidate for taxol production . The production rate was 4.7 × 10³‐fold higher than that found in the culture broth of an earlier reported fungus, Taxomyces andreanae. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of human cancer cells tested in an apoptotic assay.     

Biosynthesis of medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) by Comamonas testosteroni during cultivation on vegetable oils

Comamonas testosteroni has been studied for its ability to synthesize and accumulate medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) during cultivation on vegetable oils available in the local market. Castor seed oil, coconut oil, mustard oil, cotton seed oil, groundnut oil, olive oil and sesame oil were supplemented in the mineral medium as a sole source of carbon for growth and PHAs accumulation. The composition of PHAs was analysed by a coupled gas chromatography/mass spectroscopy (GC/MS). PHAs contained C6 to C14 3-hydroxy acids, with a strong presence of 3-hydroxyoctanoate when coconut oil, mustard oil, cotton seed oil and groundnut oil were supplied. 3-hydroxydecanoate was incorporated at higher concentrations when castor seed oil, olive oil and sesame oil were the substrates. Purified PHAs samples were characterized by Fourier Transform Infrared (FTIR) and 13C NMR analysis. During cultivation on various vegetable oils, C. testosteroni accumulated PHAs up to 78.5-87.5% of the cellular dry material (CDM). The efficiency of the culture to convert oil to PHAs ranged from 53.1% to 58.3% for different vegetable oils. Further more, the composition of the PHAs formed was not found to be substrate dependent as PHAs obtained from C. testosteroni during growth on variety of vegetable oils showed similar compositions; 3-hydroxyoctanoic acid and/or 3-hydroxydecanoic acid being always predominant. The polymerizing system of C. testosteroni showed higher preference for C8 and C10 monomers as longer and smaller monomers were incorporated less efficiently.     

Genetic instability of the short-living ascomycetous fungus <Emphasis Type="Italic">Podospora anserina</Emphasis> induced by prolonged submerged cultivation

Investigation of genetic variability of the short-living filamentous fungus Podospora anserina during its adaptation to conditions of prolonged submerged cultivation has been carried out for the first time. Cultivation of P. anserina under aeration (on a shaker) provides pronounced selective pressure, which makes it possible to obtain isolates with specific features, which are well adapted to cultivation in liquid media and have a life span several times exceeding that of the original strain. Static cultivation did not prevent the ageing of P. anserina. Repeated transfers in the shaker culture resulted in formation of mycelium deprived of the dark pigment melanin and actively producing carotenoids under illumination. The qualitative composition of P. anserina carotenoids was the same as in the closely-related species Neurospora crassa. The features obtained during the shaker cultivation (including changes in the colony morphology and decreased capacity for melanin synthesis) are inherited by their hybrids with the wild type strains, i.e., they resulted from the intragenomic rearrangements occurring during submerged cultivation of the fungus.     

液体地膜覆盖对棉花根系生长发育的影响

基于棉田可持续发展的思想,利用茚三酮法、钼蓝法及土壤双向切片法,研究了液体地膜覆盖对棉花根系生长发育的影响。结果表明,覆盖棉花前期根系生长发育加快,表现为根系活力增强,根系干重较大,但覆盖不利于棉花根系下扎,土壤深层根系衰减较快。与塑料地膜覆盖相比,液体地膜覆盖增强根系吸收与合成能力的效应在棉花各生育阶段均较明显,根系在土壤内分布较为合理,土壤深层根系衰减较慢,更有利于棉株均衡生长发育,防止棉花早衰。在棉花生产上,采用液体地膜覆盖栽培是一项可行技术．     

Utilization of substrates during batch growth of Pseudomonas fluorescens on olive oil,lard, and mutton tallow

Summary Similar amounts of cell-associated and cell-free lipase activities were present in log phase cultures of Pseudomonas fluorescens growing on fatty substrates. The rates of triglyceride hydrolysis and fatty acid utilization were balanced as free fatty acids remained at low concentrations in the culture media at all times. The time required for growth initiation and optimal growth rates of P. fluorescens were similar on olive oil, lard and mutton tallow, provided the coalesced solid fats were mechanically dispersed during the initial stages of cultivation. Although olive oil was completely consumed at the end of cultivation, substantial amounts of tri- and diglycerides, presumably bearing saturated acyl residues, remained in lard culture and, to a lesser extent, in mutton tallow culture.     

环境因子对一种病原真菌(Amoeboaphelidium sp.)感染微藻细胞能力的影响

油球藻(Graesiella sp.WBG-1)是一株适合于开放池规模化培养的产油微藻,在室外规模培养过程中常因一种病原真菌(Amoeboaphelidium sp.)的污染严重影响其生长和油脂积累,甚至导致培养失败。本文利用MTT染色法借助光学显微镜对被病原真菌感染的油球藻细胞计数并统计感染率,设置不同温度、光照强度、p H以及通气量等培养条件,研究环境因子对病原真菌感染能力的影响。结果表明:MTT染色法简单易行,可用于油球藻规模培养中病原真菌的检测;温度、光照强度、通气量和p H值均能够显著影响病原真菌的感染能力,高温、高光照强度、弱酸性环境和藻液静止等培养条件不利于病原真菌对油球藻的感染;在温度30℃、光强140μmol·m~(-2)·s~(-1)、通气量1.0 L·min~(-1)和p H 9.0±0.5的培养条件下成功建立了病原真菌感染油球藻的连续传代培养。本研究在实验室内模拟油球藻规模培养中被病原真菌感染的全过程,为深入研究该病原真菌对微藻细胞的感染行为及其感染机制提供了平台。     

Induction of laccase activity in the edible straw mushroom,Volvariella volvacea

Volvariella volvacea, strain V14, produces multiple forms of extracellular laccase when grown in submerged culture in a defined medium with glucose as sole carbon source, and on cotton waste 'compost' representative of the conditions used for industrial-scale mushroom cultivation. In liquid culture, enzyme synthesis is associated with the onset of secondary growth, and is positively regulated by copper (up to 200 microM CuSO(4)) and by various aromatic compounds. In solid-state systems, only low levels of laccase are detectable during the vegetative growth phase but enzyme activity increases sharply at the onset of fruiting and during sporophore development.     

Description of conidia from submerged cultivation of Thermomyces lanuginosus for use as a uniform inoculum.

Conidia produced by submerged cultivation of the thermophilic fungus Thermomyces lanuginosus were superior to conidia from agar plates when used as inoculum, due to a faster and more synchronous germination. With conidia derived from submerged liquid culture at 40-45 degrees C more than 90% germination was achieved at 50 degrees C within 3 h whereas the same percentage germination was only achieved after 5 h incubation of conidia produced on agar plates. The temperature during conidial formation, and conidial age at the time of harvesting, were factors influencing germination of the conidia.     

Alteration of steroid hormone sensitivity during the cultivation of human mammary carcinoma cells

Summary During early cultivation steps of the newly derived and karyotyped human mammary carcinoma line EFM-19, the cells developed faster growth rates and became increasingly less responsive to the presence of serum in the culture medium. No drastic alterations of the morphology and of the karyotype were observed, and carcinoembryogenic antigen remained expressed during the course of the cultivation. In experimental incubations at various time intervals after the explantation, the cell proliferation was analyzed for dose-dependent effects of estradiol, cortisol, progesterone, and testosterone. After 16 wk of cultivation of the stock culture in the presence of estradiol, the cells had acquired a distinct sensitivity to estradiol resulting in permanent growth enhancement. The withdrawal of cortisol from the medium of the stock culture subsequently provoked the loss of the initially noted stimulation of the proliferation by cortisol. The stimulatory effect of progesterone on the proliferation was reversed to inhibition when the stock culture was deprived of cortisol in the growth medium. The results indicate that the choice of steroid hormones in the stock culture medium was determining the quality of the cellular growth responses. This work was supported by the Deutsche Forschungsgemeinschaft, SFB34.     

Application of oil refinery waste in the biosynthesis of glycolipids by yeast

Candida antarctica or Candida apicola synthesized surfactants (glycolipids) in the cultivation medium supplemented with oil refinery waste, either with soapstock (from 5.0% to 12.0% v/v) or post-refinery fatty acids (from 2.0% to 5.0% v/v). The efficiency of glycolipids synthesis was determined by the amount of waste supplemented to the medium and was from 7.3 to 13.4 g/l and from 6.6 to 10.5 g/l in the medium supplemented with soapstock and post-refinery fatty acids, respectively. The studied yeast also synthesized glycolipids in the non-supplemented medium however, by the enrichment of medium with the oil refinery waste, a 7.5-8.5-fold greater concentration of glycolipids was obtained in the post-culture liquid then in the medium without addition of oil refinery waste. The yeast synthesized from 6.6 to 10.3 g dry biomass/l and the intra-cellular fat content was from 16.8% to 30.2%. The efficiency of glycolipids synthesis was determined by yeast species, medium acidity and culture period. The surface tension of the post-culture liquid separated from yeast biomass was reduced to 35.6 mN/m, which corresponded to the surface tension obtained at the critical micelle concentration of glycolipids.     

Lignin Peroxidases, Manganese Peroxidases, and Other Ligninolytic Enzymes Produced by Phlebia radiata during Solid-State Fermentation of Wheat Straw

The white rot fungus Phlebia radiata 79 (ATCC 64658) produces lignin peroxidase (LiP), manganese peroxidase (MnP), glyoxal oxidase (GLOX), and laccase in the commonly used glucose low-nitrogen liquid medium. However, the enzymes which this fungus utilizes for selective removal of lignin during degradation of different lignocellulosic substrates have not been studied before. Multiple forms of LiP, MnP, GLOX, and laccase were purified from P. radiata culture extracts obtained after solid-state fermentation of wheat straw. However, the patterns of extracellular lignin-modifying enzymes studied were different from those of the enzymes usually found in liquid cultures of P. radiata. Three LiP isoforms were purified. The major LiP isoform from solid-state cultivation was LiP2. LiP3, which has usually been described as the major isoenzyme in liquid cultures, was not expressed during straw fermentation. New MnP isoforms have been detected in addition to the previously reported MnPs. GLOX was secreted in rather high amounts simultaneously with LiP during the first 2 weeks of growth. GLOX purified from P. radiata showed multiple forms, with pIs ranging from 4.0 to 4.6 and with a molecular mass of ca. 68 kDa.     

Antagonistic effect of Erwinia herbicola on in vitro spore germination and germ tube elongation of Botrytis cinerea and Penicillium expansum

Interaction between Erwinia herbicola and Botrytis cinerea and Penicillium expansum was studied in liquid culture. The results show that the bacteria directly inhibited spore germination of both fungi, especially during the first hours of the paired cultivation. The distinct taxis of bacteria to spores and germ tubes was frequently followed by their lysis. It is likely that bacteria act also by competition for nutrients. The rate of antagonistic activity of the bacteria against both fungi depended on their concentration in the mixture. Formation of chlamydospore-like structures at the apical end of B. cinerea germ tube suggests induction of a defence mechanism of this fungus while in unfavourable conditions.     

Liquid-surface immobilization system and liquid–liquid interface bioreactor: Application to fungal hydrolysis

Novel fungal cultivation and bioconversion systems are proposed. Spores and mycelia of a fungus suspended in a liquid medium were effectively floated on a liquid surface by the aid of a ballooned microsphere (MS). Many fungi such as Aspergillus and Penicillium formed a thick and physically strong fungus-MS mat on the liquid surface followed by stationary cultivation (LSI). The fungus-MS mat of Absidia coerulea IFO 4423 was overlaid by a solution of 2-ethylhexyl acetate (1) in n-decane (liquid–liquid interface bioreactor, L-L IBR). The strain could efficiently catalyze the hydrolysis of 1 to 2-ethyl-1-hexanol (2). The accumulation of 2 in the L-L IBR was significantly higher than those in emulsion and organic-aqueous two-liquid-phase systems and a formerly reported interface bioreactor (solid–liquid interface bioreactor, S-L IBR). Furthermore, lipase production in the LSI system was also higher than that in a submerged cultivation system.     

Formation of an extracellular system of enzymes during growth of Geotrichum candidum 3C on cell walls isolated from cereal grain capsules

The activities of extracellular systems of hemicellulases, pectinases, and cellulases was studied during a 72-h cultivation of Geotrichum candidum 3C. The culture was grown on a medium containing 3% cell walls isolated from wheat grain capsules, which served as the sole carbon source. Enzymes catalyzing the degradation of pectin substances (beet pectin, alpha-L-arabinan, and 1,4-beta-D-galactan), as well as beta-D-galactosidase and alpha-L-arabinofuranosidase involved in their hydrolysis, were formed first (4 h after the beginning of cultivation). Enzymes hydrolyzing 4-O-methyl-alpha-D-glucurono-beta-D-xylan and sodium carboxymethyl xylan were also found in the culture liquid after 4 h of fungal growth. The contents of pectin-degrading and xylanolytic enzymes reached their maximum levels after 52-56 and 72 h of growth, respectively. Cellulolytic enzymes were detected after 8-28 h of cultivation. Enzymes degrading alpha-D-galacto-beta-D-mannan were found 24 h after the beginning of growth; their content was maximum after 72 h of cultivation.     

巴西蘑菇对木质纤维素的降解与转化*

巴西蘑菇能够降解棉籽壳和麦草两种培养基中木质纤维素复合体中的全部组分,属于白腐真菌;巴西蘑菇降解的有机物质的绝大部分被菌体的呼吸过程消耗掉,其绝对生物学效率较低,仅为4.41%~5.25%;在栽培前期木质素的降解速率大于纤维素和半纤维素,这对纤维素和半纤维素的降解十分有利;非木质纤维素组分主要在菌丝生长阶段被利用,而木质纤维素是子实体生长发育阶段的主要碳源;就整个栽培过程而言,巴西蘑菇生长发育所需要的82.39%~84.50%的碳源来自木质纤维素。     

Screening and production of rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oils

World production of oils and fats is about 2.5 million tonnes, 75% of which are derived from plants. Most of them are used in the food industry for the manufacture of different products, or directly as salad oil. Great quantities of waste are generated by the oil and fat industries: residual oils, tallow, marine oils, soap stock, frying oils. It is well known that the disposal of wastes is a growing problem and new alternatives for the use of fatty wastes should be studied. Used frying oils, due to their composition, have great potential for microbial growth and transformation. The use of economic substrates such as hydrophobic wastes meets one of the requirements for a competitive process for biosurfactant production. In the Mediterranean countries, the most used vegetable oils are sunflower and olive oil. Here we present a screening process is described for the selection of micro-organism strains with the capacity to grow on these frying oils and accumulate surface-active compounds in the culture media. From the 36 strains screened, nine Pseudomonas strains decreased the surface tension of the medium to 34-36 mN/M; the emulsions with kerosene remained stable for three months. Two Bacillus strains accumulated lipopeptide and decreased the surface tension to 32-34 mN/m. Strain Ps. aeruginosa 47T2 was selected for further studies. The effect of nitrogen and a C/N of 8. 0 gave a final production of rhamnolipid of 2.7 g l-1 as rhamnose, and a production yield of 0.34 g g-1.     

Production of ethanol by filamentous and yeast-like forms of <Emphasis Type="Italic">Mucor indicus</Emphasis> from fructose,glucose, sucrose,and molasses

The fungus Mucor indicus is found in this study able to consume glucose and fructose, but not sucrose in fermentation of sugarcane and sugar beet molasses. This might be an advantage in industries which want to selectively remove glucose and fructose for crystallisation of sucrose present in the molasses. On the other hand, the fungus assimilated sucrose after hydrolysis by the enzyme invertase. The fungus efficiently grew on glucose and fructose and produced ethanol in synthetic media or from molasses. The cultivations were carried out aerobically and anaerobically, and manipulated toward filamentous or yeast-like morphology. Ethanol was the major metabolite in all the experiments. The ethanol yield in anaerobic cultivations was between 0.35 and 0.48 g/g sugars consumed, depending on the carbon source and the growth morphology, while a yield of as low as 0.16 g/g was obtained during aerobic cultivation. The yeast-like form of the fungus showed faster ethanol production with an average productivity of 0.90 g/l h from glucose, fructose and inverted sucrose, than the filamentous form with an average productivity of 0.33 g/l h. The biomass of the fungus was also analyzed with respect to alkali-insoluble material (AIM), chitin, and chitosan. The biomass of the fungus contained per g maximum 0.217 g AIM and 0.042 g chitosan in yeast-like cultivation under aerobic conditions.